木糖利用的细胞记忆机制研究

The engineered Saccharomyces cerevisiae with high efficient xylose utilization was obtained by the adaption in xylose media. However, the capacity of xylose utilization was decreased in these strains in the conditions without xylose. This severely limited the industrial application of these engineered strains. Cellular memory was first introduced in to the research of xylose utilization. We will try to figure out the memory carriers for xylose utilization in yeast, and further reveal the inheritance modes. First, we expect to build the models of fast degradation, slow degradation and re-generation of xylose utilization capacity in engineered xylose-fermenting strain. The variance of some components including RNA, proteins, and metabolites in the three models was investigated to find the key molecule involved in cellular memory. The effect of histone modification on cellular memory was also investigated. Then it was quantitatively analyzed how the key molecular and histone modification were inherited from one cell generation to the next to predict the cellular memory mechanism of xylose utilization capacity. According to our predicted mechanism, the key genes were engineered to enhancing and/or weakening of cellular memory to accelerate the memory generation and/or stop the degradation of xylose utilization capacity. This study provides valuable insights into the rational design of complex traits in the field of biochemical engineering.

可以高效利用木糖的酿酒酵母主要是在木糖环境中强化工程酿酒酵母的木糖利用能力获得，但这类菌株的木糖利用能力在无木糖环境中有退化现象，严重限制了该类菌株的应用。申请者首次将细胞记忆概念引入木糖利用研究中，尝试发现菌株木糖利用能力变化的细胞记忆存储的物质基础，进而深入揭示木糖利用细胞记忆的遗传方式。首先建立木糖利用能力细胞记忆快速消退、慢速消退和再产生的研究模型，定性和定量分析细胞记忆与环境变化的关系；然后解析各类细胞组分（RNA、蛋白和代谢物）及组蛋白修饰在细胞记忆不同研究模型中的变化规律，挖掘细胞记忆存储的关键介导物；进而定量关键介导物在不同研究模型的子代细胞中的分配规律，建立细胞记忆传递的分子机制模型；最后调控细胞记忆分子机制模型关键节点的关联基因，强化或/和弱化细胞记忆，快速提升和固化木糖利用能力。本研究将为突破生化工程领域长期存在的微生物的环境相关复杂性状的理性改造难题提供新的思路。

项目按照计划，为了研究木糖利用的细胞记忆机制，以酿酒酵母的木糖利用体系为例展开。设计构建了不同的消耗木糖的酵母工程菌，利用代谢过程参数建立了木糖利用能力细胞记忆的过程的模型；探究了酿酒酵母木糖利用能力的细胞记忆规律，分析了不同种子培养基连续传代造成木糖利用菌株木糖利用能力的差异，在定量模型中定量阐述了传代培养过程中木糖利用能力的退化及进化；通过组蛋白修饰差异，确定了组蛋白乙酰化修饰是酿酒酵母代谢记忆的重要方式，尤其是H3K36甲基化与去甲基化修饰对细胞记忆，对乙酰化酶和关键的组蛋白位点进行了深入的分子生物学解析，推测了酿酒酵母细胞记忆的可能机制；结合微流控实时监测技术，精确刻画了酵母的木糖利用细胞记忆生成和消退。本研究为复杂生物功能的人工控制提供了新见解。